Gene therapy holds great promise as a future treatment for many different diseases. However, while over 200 therapeutic trials have been approved, few patients have demonstrated a clinical benefit. Several technical problems must be overcome before gene therapy can become a practical therapeutic alternative. One of these problems is low-level, inconsistent expression of transduced genes. In the case of gene transfer via retroviruses this is partially due to retroviral silencing. We hypothesize that chromatin structure, including DNA methylation, histone acetylation, and accessibility of trans-factors play a critical role in the silencing and expression of retrovirally transduced genes. Our specific aims are: 1) to characterize the role of chromatin structure in long term silencing; and 3) to determine the utility of pharmacologic agents in the reversal and prevention of retroviral silencing in hematopoietic cells. We have established a model of retroviral vector silencing in clonal populations of hematopoietic cells. We propose to use this model system to comprehensively characterize the changes in chromatin structure that occur during retroviral vector silencing and the ability of drugs which modulate chromatin structure to inhibit or reverse silencing. It is our hope that these experiments will lead to strategies to achieve consistent, high- level expression from integrated gene therapy vectors.